Cerium and samarium blocked antioxidant enzymes in wheat plants

This work was conducted to study positive and negative impacts of cerium (Ce) and samarium (Sm) on two cultivars (Arta and Baharan) in wheat plant. Symbols of stress such as proline, malondialdehyde (MDA) and antioxidant enzymes, which may be complicated in the suppression responses of plants, were also studied. Wheat plants were exposed to 0, 2500, 5000, 7500, 10,000 and 15,000 μM of Ce and Sm for 7 days. The growth enhanced in plants treated with lesser Ce and Sm concentration (2500 μM) and declined in plants treated with upper concentrations as compared to untreated plants. The treatment with 2500 μM of Ce and Sm increased dry weigh in Arta by 68.42 and 20% and in Baharan by 32.14% and 27.3%. Thus, Ce and Sm had hormesis effect on growth in wheat plants. According to plant’s growth parameter patterns, Arta cultivar had more sensitive to Sm than to Ce, whereas Baharan cultivar had sensitive to Ce than to Sm. Our results indicated impact of Ce and Sm on proline accumulation depended on the dosage of Ce and Sm. It was observed that Ce and Sm accumulated in wheat plants at higher exposure doses. Increment of MDA content by Ce and Sm treatments showed that these metals caused oxidative stress in wheat plants. Ce and Sm blocked enzymatic antioxidant system (superoxide dismutases, peroxidase and polyphenol peroxidase) in wheat. In wheat plants treated with lower Ce and Sm concentrations higher amounts of non-enzymatic antioxidant metabolites were detected. Thus, we showed the potential negative impact of unsuitable utilization of REEs in plants and suggested growth and interruption in physiological and biochemical mechanisms as a possible factor to recognize the underlying toxicological processes.

www.nature.com/scientificreports/ include catalase (CAT), superoxide dismutases (SOD), ascorbate peroxidase (APX) and peroxidase (POX) and non-enzymatic ones, such as phenolic compounds, ascorbate, glutathione and tocopherol 10 . The enhancement of flavonoid production and phenolic compounds is one of defense strategies in plants against the stress caused by REEs 11 . Wheat (Triticum aestivum L.) is one of the chief sources of protein and calories and one of the main cereals in the world. About 82% and 85% of the worldwide population relates with wheat for protein and basic calories, respectively 12 . Also, this plant is applied in the production of different types of wheat products, such as steamed breads, flat, leavened bread, cakes, biscuits, pasta and noodles. Beyond its application for human utilization, wheat is also used for the increase of non-edible products such as fuel. Production of wheat is severely influenced by adverse environmental conditions 13 . Wheat is an experiment crop because it is one of the key economic plants and is cultivated on a universal scale. Moreover, wheat is commonly used such as an ecotoxicological pointer 14 .
Barely any data is available about the impact of Sm on growth and antioxidant response in wheat plant. In this regard, the purpose of this present work was to study the effects of Sm and Ce on growth in wheat plant. Aiming to better comprehend the behavior of this plant in the presence of two REEs, tolerance ability to these elements was evaluated. Also, antioxidant systems include enzymatic and non-enzymatic, proline accumulation, MDA content such as oxidative stress indicator and hydrogen peroxide (H 2 O 2 ) content of wheat plant were assessed to determine the biological functions and physiological processes of wheat to REEs exposure. This work will involve to a deeper empathetic of the negative impacts caused by extreme treatment of REEs.

Results
Biomass and accumulation of proline. After 21 days of Ce or Sm exposure, the growth parameters of the wheat plants showed significant alternations when treated with various concentrations of REE (Tables 1, 2). Plant FW increased significantly by 56.43% in Arta and 25.4% in Baharan at 2500 µM Ce treatment, respectively, as compared to control. Other levels of Ce treatment declined plant FW in two cultivars and the rate of reduction was the highest levels in 15,000 µM Ce-treated plants. Ce treatment at 2500 µM enhanced (68.42%) plant DW in  30.30% for 10,000 μM and 45.45% for 15,000 μM. In Arta cultivar, the plant height and root length were not significantly affected by the application of 2500-7500 µM Sm, compared with the control. On the other hand, the application of 10,000 and 15,000 μM Sm reduced the plant height and root length in Arta cultivar in comparison with the control. Conversely, the shoot length in Arta cultivar showed a significant trend with the application of Sm. The plant height and root length in Baharan cultivar did not change significantly at Sm concentrations up to 10,000 µM, while these parameters dropped with highest level of Sm (15,000 µM). Also, no significant difference was found in the shoot length between the Sm-treated and control plants in Baharan cultivar ( Table 2).
No significant impacts on proline content were detected in all treatment of Ce in Arta cultivar, except for plants treated with 15,000 µM Ce. The 15,000 µM Ce treatment enhanced proline accumulation by 844 fold in Arta cultivar. In Baharan cultivar, low and moderate concentrations of Ce (2500-7500 µM) had no significant impact on proline content as compared to that of control, whereas high Ce concentrations (10,000 and 15,000 µM) caused a significant rise by 54.2 and 101.2 fold in proline content, respectively (Table 3). Proline content was not significantly affected by 2500-7500 µM Sm treatment in Arta cultivar relative to that of the control treatment. The exposure of 10,000 and 15,000 µM Sm caused significant elevation by 16 and 33.12 fold of proline content in Arta cultivar, displaying an increasing trend with exposure dose. Treatments with 7500 µM Sm in Baharan cultivar induced a significant enhancement in proline content, whereas other treatments did not cause any significant impact on proline content (Table 4).  (Table 3). Application of varying Sm doses to Arta and Baharan cultivar significantly declined H 2 O 2 content and maximum decrease showed at 15,000 µM ( Table 4).
The 2500 µM Ce treatment did not cause any significant impact on MDA content in both cultivars. The MDA content at 5000, 7500, 10,000 and 15,000 µM Ce treatment was 114.28, 265.71, 258.57 and 285.71% in Arta cultivar and 75, 125, 211.25, 200% in Baharan cultivar larger than the control (Table 3). Treatment with 2500 µM Sm had no significant effect on the MDA content in both cultivars. The MDA content showed a significant rise  (Table 4).

Enzymatic antioxidant system.
Ce treatment boosted protein content in both cultivars (Fig. 1a). Sm treatment also caused an increase in the protein content in Arta cultivar, whereas this was not caused significant change trend in Baharan cultivar (Fig. 1b).
The activities of the ROS scavenging enzymes such as SOD, POX and PPO are shown in Figs. 1, 2. Ce treatment considerably declined SOD activity in both cultivars as compared to that of control and no significant difference was found in the SOD activity between various levels of Ce. Reduction in SOD activity of Baharan Cultivar was more than Arta cultivar (Fig. 1c). No significant difference was found between the POX activity in Ce-treated and control plants in Arta cultivar. A decrease was seen in POX activity of Baharan cultivar under Ce treatment as compared to control (Fig. 2a). PPO activity was significantly reduced in two cultivars at all Ce doses (Fig. 2c).
All of Sm treatments dropped SOD activity in both cultivars, but this reduction was higher in Arta cultivar when compared to Baharan cultivar (Fig. 1d). A decrement in POX activity occurred in all levels of Sm treatment in two cultivars and no significant changes in POX activity were observed between different concentrations (Fig. 2b). PPO activity enhanced in Arta cultivar by 2500 µM Sm and then decreased at higher concentrations.  (Fig. 2d).

Non-Enzymatic antioxidant system. The effect of Ce and Sm on the non-enzymatic antioxidants of two
wheat cultivars is displayed in Figs. 3, 4. Total phenol content was heightened under Ce treatment up to 2500 µM in both cultivars; the effect of Ce on total phenol content was more pronounced in Baharan cultivar (Fig. 3a). Flavonoid content in both cultivars boosted in plants treated with 2500 µM Ce, but in the plants treated with other doses of Ce a significant decline was observed (Fig. 3b). Anthocyanin content only enhanced by 2500 µM Ce whereas it reduced in both cultivars at 5000-15,000 µM Ce as compared to non-treated plants (Fig. 4a).
The total phenol content in Arta cultivar was significantly reduced and lower than that of the control under Sm treatments. Total phenol content was higher in Baharan cultivar exposed to 2500 and 5000 µM Sm comparison with the control, whereas higher doses caused significant decrease in this content (Fig. 3b). A significant decrease  www.nature.com/scientificreports/ in flavonoid content in Arta cultivar was detected under Sm treatments. Treatment with 7500-15,000 µM Sm declined flavonoid content in Baharan cultivar, while no significant differences were found at other Sm treatments, compared with the control (Fig. 3d). Similar results were observed for anthocyanin content in both cultivars under Sm treatment. In both cultivars, anthocyanin content increased at 2500 and 5000 µM Sm, then decreased up to 15,000 µM Sm to higher values than that of control (Fig. 4b).

Investigation of correlation between various Ce and Sm levels and studied parameters by PCA analyze. The potential correlations among the studied variables under the different treatments of Ce and
Sm were analyzed based on Pearson's correlation coefficient. According to this analyze, DW or growth in wheat plants treated with Ce and Sm had more positive correlation with non-enzymatic antioxidant system than that enzymatic antioxidant system. Thus, these findings confirmed that that non-enzymatic antioxidant mechanism is more important for cope on destructive effects of Ce and Sm in wheat plants. In both treatments, growth negatively correlated with MDA level. The control treatment adjudged as the best value giving treatments. Its  www.nature.com/scientificreports/ impact was followed by that of 2500 µM of Ce and Sm. This displayed the stimulatory effect of 2500 µM. The other doses caused growth limitations in wheat plants as these treatments were clustered on the other side of loading plot (Fig. 5a,b).

Discussion
Numerous studies have revealed that the elevation of growth and development in plants happens under low levels of REEs, and an inhibitory impact has been witnessed under high levels 15,16 . Our results indicated that the negative or positive impacts of Ce and Sm on growth depended on the dosage of Ce and Sm. We found that two cultivars did not show any signs of damage and that the growth of the plants enhanced when the Ce and Sm concentration was 2500 µM. However, when the Ce and Sm concentration exceeded 2500 µM, the growth of two cultivars was decreased and significantly lowers than those of the control group. These findings indicated that Ce and Sm had a hormesis impact on growth in wheat plants. Based on our results, negative effect of Ce and Sm was more on the root than on the shoot. Also, our findings recommended that two cultivars can show a different sensitivity to Ce and Sm. Arta cultivar was more resistant to Ce, but Baharan cultivar was more resistant to Sm. In our work, appropriate concentrations of Ce (2500 µM) and Sm (2500 µM) effectively enhanced the non-enzymatic antioxidant system of both cultivars and declined the negative effect of Ce and Sm, and subsequently increased growth. However, high concentrations of Ce and Sm blocked enzymatic antioxidant system and induced oxidative damage, and severely depressed the plant growth. Ce affect metabolism in Lemna minor L. with a biphasic trend, with stimulatory impacts at lesser levels and inhibitory impacts at upper levels 17 . The stimulated growth in REEs-treated plants is directly correlated to cell division 18 , enhancement of mitotic rate 19 and augmentation of the absorption of valuable ions for plants 20 . However, study of some molecular parameters and the genes involved in oxidative stress resistance in reaction to Ce and Sm poisonousness, would aid to more understand of adaptation strategies adopted by this plant to decrease Ce and Sm stress. Proline accumulates in plants in response to water, salinity and heavy-metal stresses. The enhancement of proline content by plants is a defensive strategy. Proline acts such as a source of carbon and nitrogen, ROS scavenger, osmotic buffer, osmoprotectant and membrane preservative 21 . The impacts of Ce and Sm on the proline content of wheat plants follow a dose-depending response with an induction at high concentrations and a reduction at low concentrations. This biphasic impact of Ce and Sm on proline accumulation is consistent with data published for Vigna unguiculata 22 . REEs are recommended to be complicated in enhancing the water consumption efficiency in plants by inducing their proline amount. Accumulation of proline in plants treated with Ce may involve to steadying subcellular components and osmotic equilibrium in the cell. The more content of proline detected in plants treated with higher levels of Ce, can be connected with enhance ability of suppressing ROS and defending cell against oxidative injury as a defense strategy 23 .
High doses of REEs may lead the ROS production and cause to oxidative stress 24 in plants. Antioxidant enzymes such as SOD, PPO and POX play a main function in the protective responses of plants. SOD activation is first line of protection against ROS under stresses, which changes superoxide anion to H 2 O 2 25 , while POX enzyme converts H 2 O 2 to O 2 and H 2 O. The specific enzymes do not react similarly to all metals. Several elements cause to an enhance in enzymes activities, whereas other elements decline the enzymes activities 26 . The excess ROS can cause lipid peroxidation (assayed as MDA content) by demeaning polyunsaturated fatty acids. The observed changes of lipid peroxidation and antioxidant system are typical indicators of REEs toxicity 27 . In our experiment, Ce and Sm treatments reduced enzymatic antioxidant system (SOD, POX and PPO) were concomitant to increased levels in lipid peroxidation (MDA content) in both cultivars, which can be regarded as typical indicator of cellular injury. Therefore, lipid peroxidation as well as disturb of antioxidant systems, i.e. usual indicators for stress conditions, happened after treatment with Ce and Sm in wheat plants. The increase in MDA level was associated with reduction in growth, thus confirming the potential danger in wheat plants. Our data proposed that the scavenging roles of POX and SOD were impaired. Chen et al. 11 suggested that the declined activity of SOD at upper Ce concentrations was incompetent to remove superoxide anion leading to boosted membrane hurt. Also, the higher dose of Ce treatment, the less effective enzymes were at suppressing ROS and the more injury to the cells.
Environmental stresses, comprising metallic stress, could augment production non-enzymatic antioxidants in plants to decline the generation of ROS and escape cell injury. Low doses of Ce and Sm induced non-enzymatic antioxidants included phenol, flavonoid and anthocyanin content, while high doses of these elements reduced accumulation of metabolites. Thus, the accumulation of non-enzymatic antioxidants was significantly dependent on the dose of Ce in wheat plants in this work. These observed alterations in non-enzymatic antioxidants might be a self-protection strategy to mitigate the metallic stress in wheat plants. Many investigators have illuminated the antioxidant ability of flavonoids and phenols in chelating uptake elements and suppressing ROS by transmitting electrons to free radicals. Metals have the capacity to decline lipid hydroperoxide (LOOH) by the hemolytic breakdown of the (O-O) bond, subsequent in lipid alkoxyl radicals and the formation of free-radical chain oxidation. Flavonoids and phenolic compounds can trap lipid alkoxyl radicals to prevent lipid peroxidation 28 . Our data are in agreement with the work of Dridi et al. 22 , where Ce-treated Helianthus Annuus plants showed a remarkable accumulation of flavonoid and phenolic compound. PAL is a main enzyme which controls phenylalanine biosynthesis into phenolic compounds 30 . Chen et al. 11 showed that the maximum PAL activity was detected at moderate concentrations of Ce (0.5-1.0 mM) and was agreement with the enhancement of flavonoid content in Ginkgo suspension cells. Thus, REEs can increase phenolic compounds by increasing PAL activity. Also, REEs enhance the generation of secondary metabolites by promoting the transcriptions of critical biosynthetic genes 31 .

Conclusion
Ce and Sm influenced growth in wheat plants following a biphasic trend, with stimulatory impacts at lower doses and inhibitory impacts at higher doses. High doses of Ce and Sm induced proline accumulation in wheat plants.
Ce and Sm treatments blocked antioxidant enzymes and enhanced lipid peroxidation. Change in non-enzymatic antioxidant compounds was detected in wheat plants under Ce and Sm exposure, with a concentration-dependent trend. Although the complete mechanism by which Ce and Sm are taken up by plants residues to be studied, we have presented some novel insights into the physiochemical mechanisms of plants in a Ce-enriched environment or Sm-enriched environment beyond mere accumulation investigates. Furthermore, future investigations will be required to recognize the molecular processes responsible for the biochemical and physiological reactions perceived.

Materials and Methods
Plant material and experimental plan. All  Growth parameter and proline content. The plants were evaluated one week after being exposed to Cerium and Samarium in terms of fresh weight (FW), dry weight (DW) and other parameters. For the proline content assessment according to Bates et al. 32 , 0.1 g of leaf tissue was homogenized in 3 mL sulfosalicylic acid (3%) and then was centrifuged at 13,249×g for 20 min. Then, 0.5 mL of supernatant was combined with acid ninhydrin (1 mL) and glacial acetic acid (1 mL) and then was boiled at 100 °C for 1 h. The reaction mixture was extracted with 2 mL toluene and the absorbance was assayed at 520 nm via l-proline as a standard. Lipid peroxidation. To assay the lipid peroxidation, a testosterone (TBA) test, which identifies MDA as the final product of lipid peroxidation was conducted according to Heath and Packer 33 . Leaf tissue (0.5 g) was homogenized in 0.1% TCA and then was centrifuged at 7840×g for 10 min. The supernatant (0.5 mL) was added to 1 mL of thiobarbituric acid (0.5%) in 20% TCA. The mixture was heated in 95 °C for 30 min and then was centrifuged at 7840×g for 10 min. The absorbance of supernatant was assayed at 532 and 600 nm.

Measurement
Enzymatic antioxidants and protein content. Leaf material (0.5 g) was extracted at 4 °C with 1 M Tris-HCl (pH 6.8) to estimate different enzyme activities. The homogenate was centrifuged at 13,249×g for 20 min at 4 °C and the obtained supernatant was kept at − 70 °C and later used for enzyme assays. Protein content was estimated based on Bradford 35 method. The superoxide dismutase (SOD) activity was measured according to Giannopolitis and Ries 36 method. Reaction solution comprised potassium phosphate buffer (50 mM), 0.1 mM EDTA, 75 μM NBT, 13 mM methionine, 75 μM riboflavin and 100 μL of enzyme extract. The reaction solution was placed in front of the light for 18 min and absorbance was assayed at 560 nm.
The activity of polyphenol oxidase (PPO) was determined according to Raymond et al. 38 . The reaction mixture included 2.5 mL of 200 mM potassium phosphate buffer (pH 6.8), 0.2 mL of 20 mM pyrogallol and 20 μL enzyme extract. The enzyme activity was assayed at 430 nm.
Non-enzymatic antioxidants. In order to preparation of methanolic extract, 0.1 g of dry tissue was homogenized in 5 mL methanol 80% and then was centrifuged at 5000×g for 20 min. For the total phenol content measurement, 0.1 mL methanolic extract was added with 2.5 mL Folin-Ciocalteu reagent 10%. The mixtures were neutralized by sodium bicarbonate 7% and then absorbance was measured at 765 nm 39 .
Content of flavonoid was determination by Chang et al. 40 method. In this method 0.1 g of leafs was homogenized in 2 mL of methanol 80%. Methanolic extract (0.5 mL) was mixed with 1.5 mL of methanol 80%, 0.1 mL of aluminium chloride (10%), 0.1 mL of potassium acetate (1 M) and 2.8 mL of distilled water and the absorbance was assayed at 415 nm after 30 min.
The content of anthocyanin was measured in 0.3% HCl in methanol at 25 °C using the extinction coefficient (33 cm 2 mol -1 ) at 550 nm 41  www.nature.com/scientificreports/ Statistical analyses. Each experiment was repeated three times and the data were analyzed by either oneor two-way analysis of variance (ANOVA) using SPSS (ver. 26). Means were compared by Duncan's test at the 0.05 level of confidence. Principal component analysis (PCA) analysis was used for obtaining correlation matrix, giving the Pearson's correlation coefficients between each pair of variables, i.e. the analytical parameters tested. PCA analysis was done by XLSTAT (2016).

Data availability
All data generated or analyzed during this study are included in this published article.